Hydrogenase enzymes are pervasive, being found in bacteria, archaea, and some higher organisms. Hydrogenases are found in many pathogens, including some that inhabit the human gut. The [FeFe] and [NiFe]-hydrogenases mediate the most fundamental chemical reaction: the interconversion of H2 with protons and reducing equivalents. Since H2 is an unusual substrate, the enzymes are also structurally exceptional with an array of distinctive cofactors, especially the site of H2 binding and release. Similarly, the biosynthesis of these active sites involves elaborate and novel biochemistry. In addition to biophysical approaches, elucidation of these mechanisms relies on organometallic chemistry, especially since the substrates (H2, H+, H-) are often invisible to conventional biophysical methods. This project aims to elucidate the biosynthesis and mechanism of action of the [FeFe] enzymes, the faster of the two main hydrogenases and the one most amenable to development for other applications. The work involves synthesis of proposed intermediates, spectroscopic and electrochemical characterization, in vitro assays, and isotopic labeling. It also relies on extensive collaborations with groups that offer specialized skills in theory, synchrotron-based spectroscopy, spin resonance, and in vitro testing. This project specifically focuses on the recently confirmed azadithiolate (adt) cofactor, which is the enzyme's most remarkable component from a mechanistic perspective. Although the adt cofactor is unstable in the free state, methods are being developed to stabilize it in protected form. It will be isotopically labeled and incorporated into an apo enzyme. These experiments will allow us to identify the precursor to the adt (main hypothesis: radical SAM induced reactions of cysteine-Fe conjugates). Prior to construction of the Fe2(adt) ensemble, two Fe-cysteine-CO centers are supposed to be preorganized in a scaffold protein HydF. This work will produce the first Fe-cysteine-CO complexes for testing in vitro and by chemical methods. Finally, the work will evaluate the role of adt on the catalytic mechanism. Of specific interest is the influence of the protonation state of the amine cofactor on the behavior of the [2Fe] subunit. The latter studies will be conducted on model complexes containing Fe2 with authentic and modified amine cofactors. Of specific interest is the influence of N-protonation on the binding of H2 and the inhibitors CO and formaldehyde. Overall, the work focuses on understanding the biosynthesis and mechanism of action of the fastest catalysts known for producing H2 from water.